Small molecules that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biology research and as clinical anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, we introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biology, and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.

Infection by HTLV-1 has been correlated with the appearance of various proliferative or degenerative diseases. Some of these disorders have been observed in transgenic mice expressing the Tax protein, which is known to transactivate various viral and cellular promoters through interactions with several transcription factors. In this study we show that the C-terminus of this viral oncoprotein represents a motif permitting binding of Tax to the PDZ domains of several cellular proteins. A two-hybrid screen with Tax as bait indeed yielded complementary DNAs coding for six proteins including PDZ domains. Two of them correspond to truncated forms of the PSD-95 and b1-syntrophin proteins, another clone codes for a protein homologous to the product of the C. elegans gene lin-7. The other three clones code for new human members of the PDZ family of cellular proteins. The interaction of Tax with the products of these clones was con®rmed by immunoprecipitation assays in mammalian cells, and analysis of various mutants of Tax established the importance of the Cterminal amino acids for several of these interactions. These data suggest that Tax could perturb the normal function of targeted cellular proteins by strongly interacting with their PDZ domains.

Activation of the HTLV‐I promoter by the viral Tax1 transactivator is mediated by a 21 bp sequence motif imperfectly repeated three times and composed of three exactly conserved domains (A, B and C from 5′ to 3′). We show here that the Tax1 response requires the integrity of the B domain and of at least one of the flanking A or C domains. We have identified three cellular proteins which bind specifically to the 21 bp motif. One of these is the already well‐characterized transcription factor ATF. The other two, namely HEB1 and HEB2, are specific for the 21 bp motif. HEB1 can bind to either domain A or C, but binding of ATF and HEB2 is determined by domain B. However, neither domain B alone, nor ATF/CREB binding sites respond significantly to Tax1. We therefore propose that Tax1 induction of the 21 bp enhancer element requires interaction with the two different cellular proteins identified in this study: HEB1 and HEB2, rather than binding of the ATF factor.

The Tax transactivator of the human T cell leukemia virus type I (HTLV-I) exhibits oncogenic properties. A screen for proteins interacting with Tax yielded a complementary DNA (cDNA) encoding the human Int-6 protein. In mice, the Int-6 gene can be converted into a putative dominant negative oncogene after retroviral insertion. Here, Int-6 was localized in the cell nucleus to give a speckled staining pattern superposed to that of the promyelocytic leukemia (PML) protein. The binding of Tax to Int-6 caused its redistribution from the nuclear domains to the cytoplasm. Thus, Int-6 is a component of the PML nuclear bodies and Tax disrupts its normal cellular localization by binding to it.

In this report, we analyzed whether the degradation of mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was affected in human T-lymphotropic virus type 1 (HTLV-1)-infected cells. This pathway was indeed strongly inhibited in C91PL, HUT102, and MT2 cells, and such an effect was also observed by the sole expression of the Tax protein in Jurkat and HeLa cells. In line with this activity, Tax binds INT6/EIF3E (here called INT6), which is a subunit of the translation initiation factor eukaryotic initiation factor 3 (eIF3) required for efficient NMD, as well as the NMD core factor upstream frameshift protein 1 (UPF1). It was also observed that Tax expression alters the morphology of processing bodies (P-bodies), the cytoplasmic structures which concentrate RNA degradation factors. The presence of UPF1 in these subcellular compartments was increased by Tax, whereas that of INT6 was decreased. In line with these effects, the level of the phosphorylated form of UPF1 was increased in the presence of Tax. Analysis of several mutants of the viral protein showed that the interaction with INT6 is necessary for NMD inhibition. The alteration of mRNA stability was observed to affect viral transcripts, such as that coding for the HTLV-1 basic leucine zipper factor (HBZ), and also several cellular mRNAs sensitive to the NMD pathway. Our data indicate that the effect of Tax on viral and cellular gene expression is not restricted to transcriptional control but can also involve posttranscriptional regulation. Human T-lymphotropic virus type 1 (HTLV-1) infection is associated with the onset of severe diseases, mainly adult Tcell leukemia (ATL) and tropical spastic paraparesis, also named HTLV-1-associated myelopathy, in 2 to 5% of patients (for a review, see reference 44). These conditions are characterized by a long latency, with infection often occurring in childhood and disease development at an adult age. Accordingly, it is estimated that the development of ATL involves several phases, ending in the acute proliferation of monoclonal ATL cells. At the initial stage, lymphocytes are infected by viral particles, leading to provirus integration and the expression of various viral proteins. Among them, Tax plays an important role both by inducing the transcription of the provirus and by stimulating the proliferation of the host cell. Tax, which is present in both the nucleus and the cytoplasm, exerts these functions by directly binding or by modulating the expression of several key cellular proteins involved in transcriptional control, cell cycle progression, genomic stability, cell adherence and migration, protein degradation, and RNA metabolism (7).Among these various cellular proteins bound by Tax, we have previously characterized INT6, also known as EIF3E, one the 13 subunits of the translation initiation factor eukaryotic initiation factor 3 (eIF3) (16). The complex between both proteins was found to be cytoplasmic, whereas in normal cells, INT6 is present in both the cytoplasm and the nucleus (16,27,64). In mammalian cell...

The viral Tax protein, which is encoded by human T-cell leukaemia virus HTLV-I, activates nuclear translocation of the NF-kappa B/Rel transcription factors and relieves cytoplasmic sequestration of RelA and Rel by heterodimerization with NF-kappa B1/p1O5 (refs 1,2). Proteolytic maturation of this precursor protein is performed by the proteasome complex. Here we show that Tax binds specifically to two subunits of the 20S proteasome, HsN3 and HC9. This interaction is weakened with HsN3 and lost for HC9 when a mutant of Tax is substituted that is selectively defective for NF-kappa B activation. Immunoprecipitation shows that p1O5 binds weakly to HC9 and that this interaction is reinforced by Tax. No bridging function of Tax between p1O5 and HsN3 was observed. From these results, we propose that Tax accelerates the proteolytic maturation of P105 by favouring its anchorage to the proteasome.

Recombinant plasmids containing mutant or wild-type adenovirus serotype 2 Ela genes that produce the 12S mRNA alone, the 13S mRNA alone, or both mRNAs were cotransfected into HeLa cells with plasmids containing the viral Ella or EIII transcription units. The amount of RNA produced from the Ella and EIII promoters was increased by the products of both the 13S and the 12S RNAs. By measuring the level of specific transcription in nuclei isolated from transfected cells we directly demonstrate that the increased amount of Ella RNA is due to stimulation of the rate of transcription.Products of the adenovirus immediate early transcription unit Ela are required for efficient expression of the other early transcriptional units, EIb, Ella, ElIb, EIII, and EIV (1, 2). Evidence for the role of the Ela gene comes from analysis of Ela mutant viruses (1-3) and from the use of inhibitors of protein synthesis during virus infection (4-6). These studies have suggested that products of the Ela gene may act by relieving the effect of a host repressor of adenovirus transcription (4-6) or by catalyzing the assembly of stable early transcription complexes (3). Recently, transient expression of cloned early viral genes has been studied after cotransfection into cultured cells (7-11) or microinjection into Xenopus oocytes (11) or cultured cells (12). The results further demonstrate the dependance of early gene expression on the presence of the Ela gene products. However, it has not been unequivocally established that the regulation takes place at the transcriptional level, since RNA transcription rates were not determined. Furthermore it is not known which of the two early Ela mRNAs, the 12S or the 13S (see Fig. 4), or both, encodes the stimulatory activity.We have examined the effect of the Ela products on Ella and EIII expression by cotransfecting into HeLa cells their cloned transcription units with recombinants coding for either the 12S or 13S mRNAs or for both. S1 nuclease analysis was used to measure the overall cytoplasmic accumulation of specific transcripts, and specific in vitro transcription in isolated nuclei was carried out to evaluate the transcription rate of the corresponding RNAs. MATERIALS AND METHODS HeLa cells grown in monolayer were transfected by the calcium phosphate precipitation technique as described (13) with 10-20 ,g (see figure legends) of recombinant DNA per 10-cm Petri dish. After 36-60 hr, cytoplasmic RNA was purified from cells lysed with 0.3% Nonidet P-40. Nuclease S1 mapping was carried out as published (14). For transcription experiments with isolated nuclei, nuclei were prepared as described (15). RESULTSAccurate in Vivo Initiation of Transcription from Cloned Ela, Ella, and EIII Transcription Units After Transfection into HeLa Cells. Plasmids containing the Ela, Ella, and ETIT transcription units (respectively pEIASV, pEll, and pElIl) were constructed as described in Fig. 1. These recombinant plasmids were used to transfect HeLa cells and the amount of specific RNA produced was measured by...

The human T‐cell leukemia virus type I (HTLV‐I) codes for the potent transcriptional activator, Tax1, which induces the enhancer activity of various enhancer elements. In the case of the 21 bp enhancer of the HTLV‐I provirus, this induction is correlated with the association of Tax1 with this DNA element via a specific cellular factor. That the indirect association of Tax1 with DNA can lead to transcriptional activation has also been supported by the study of chimeric GAL4‐Tax1 proteins. The GAL4‐Tax1 stimulatory effect exhibits a strong self‐squelching. In order to determine whether Tax1 interacts directly with the general transcription factors or via intermediary molecules, we have analyzed how overexpression of the TATA binding protein (TBP) and TFIIB protein affects the squelching curve of GAL4‐Tax1. The data presented here show that overexpression of TBP strongly increases the stimulatory effect of GAL4‐Tax1, causes a displacement of the maximum of the squelching curve and partially alleviates the squelching. Under similar conditions TFIIB exhibited little effect. From these results we conclude that Tax1 can increase the recruitment of TBP by directly interacting with this protein. Biochemical experiments with purified proteins produced in bacteria confirmed that Tax1 can interact with TBP but not with TFIIB. Tax1 interacts with the conserved C‐terminal part of TBP. Analysis of the ability of different mutants of Tax1 fused to the GAL4 DNA binding domain to activate transcription and to associate with TBP, showed that these activities are correlated. However, since one transcriptionally inactive mutant was able to interact efficiently with TBP in vitro, it would appear that an event other than the Tax1‐TBP contact also intervenes in the activation of transcription by Tax1.